Method for detecting SARS coronavirus

ABSTRACT

This invention provides: a method for detecting SARS pathogenic viruses with high sensitivity and rapidity for diagnosing severe acute respiratory syndrome (SARS); an oligonucleotide primer that can specifically hybridize with any nucleotide sequence constructed based on the nucleotide sequence of RNA polymerase of the SARS coronavirus; a method for nucleic acid amplification using such primer; a method for diagnosing infection with the SARS coronavirus via detection of nucleic acid amplification; and a kit for diagnosing SARS.

This is a divisional of U.S. patent application Ser. No. 10/561,947 nowU.S. Pat. No. 7,399,588 B2, filed Dec. 22, 2005, which is the NationalStage of PCT/JP04/08355 filed Jun. 15, 2004, and which claims priorityfrom Japanese Application No. 2003-184123 filed Jun. 27, 2003 andJapanese Application No. 2003-384572 filed Nov. 14, 2003. The entiredisclosure of the prior application is hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to a method for detecting the severe acuterespiratory syndrome (SARS) coronavirus and more particularly to amethod for diagnosing SARS via a highly sensitive method for detectinggenes.

BACKGROUND ART

Severe acute respiratory syndrome (hereinafter abbreviated as “SARS”) isan infectious disease that began in Guandong, China in Nov. 2002, andhas caused serious infection in nations such as Hong Kong, Taiwan, andCanada. According to the World Health Organization (WHO), the mortalityfor the patients afflicted with SARS is deduced to be 15% on average,and it is deduced to be 50% or higher in the case of patients aged 65and over. The SARS coronavirus, which is a pathogenic virus of SARS, isa single-strand RNA virus (see, for example, non-patent document 1). Itis known that this virus infects animals other than humans.

Major clinical symptoms of SARS are fever with temperatures of 38° C. orhigher and respiratory problems, such as coughing and difficulty ofbreathing. In some cases, symptoms such as headache, shaking chills,loss of appetite, generalized malaise, diarrhea, or clouding ofconsciousness are observed. However, these symptoms are almost the sameas those of other respiratory diseases, such as influenza. Thus, it isdifficult to distinguish SARS from other diseases based solely on itssymptoms.

An immunologic procedure has been known as a method of clinical testing.In such testing, the presence of an antibody against a viral antigen inblood, serum, urine, or saliva is inspected. The enzyme-linkedimmunosorbent assay (ELISA) and the immunofluorescence assay (IFA) areknown techniques for detecting antibodies against the SARS coronavirus.With these techniques, however, antibodies cannot be detected at theearly stage of the disease. In the case of ELISA, antibodies cannot bedetected until 20 days after the development of the disease. In the caseof IFA, antibodies cannot be detected until 10 days after thedevelopment of the disease (see, for example, non-patent document 2).

Also, a method for detecting antibodies via amplification of the virusgene via PCR has been known. This technique, however, has beenproblematic since it takes 1 hour or longer for amplification anddetection, and the detection sensitivity thereof is low. Accordingly, amethod for detecting the SARS coronavirus with rapidity and highsensitivity has been awaited (see, for example, non-patent documents 3and 4).

The present inventors found that the aforementioned problems could besolved by the LAMP method, which is a method capable of detecting theSARS coronavirus with higher sensitivity and specificity within ashorter period of time compared with conventional techniques, such asimmunoassay or PCR. Thus, the present inventors attained the object ofthe present invention.

[Non-Patent Document 1]

The World Health Organization Update 49-SARS case fatality ratio,incubation period, 7 May 2003, Case fatality ratio (date of search: Jun.24, 2003),

[Non-Patent Document 2]

SARS: a method of diagnostic assay (April 29, Revision 4-1), date ofsearch: Jun. 24, 2003, the Infectious Disease Surveillance Center(IDSC), the National Institute of Infectious Diseases

[non-patent document 3]

Drosten C., et al., New Eng. J. Med., 2003, vol. 348, pp. 1967-1976

[Non-Patent Document 4]

“Detection of SARS coronavirus gene via RT-PCR (date of renewal: May 16,2003), date of search: Jun. 24, 2003, the Laboratory of InfluenzaViruses, Department of Virology Iii, the Infectious Disease SurveillanceCenter (IDSC), the National Institute of Infectious Diseases

SUMMARY OF THE INVENTION

It is an object of the present invention to detect a pathogenic virus,i.e., the SARS coronavirus, with high sensitivity for early diagnosis ofSARS.

The present inventors have conducted concentrated studies in order toattain the above object. As a result, they have found that the SARScoronavirus could be detected with high sensitivity by producing anoligonucleotide primer that can selectively hybridize with a SARScoronavirus-specific nucleotide sequence and amplifying a SARScoronavirus-specific nucleotide sequence by the LAMP method. This hasled to the completion of the present invention.

Specifically, the present invention includes the following elements.

(1) An oligonucleotide primer designed based on any nucleotide sequenceselected from nucleotides 41 to 256 of the nucleotide sequence of an RNApolymerase of the SARS coronavirus as shown in SEQ ID NO: 1 or anucleotide sequence complementary thereto.

(2) The oligonucleotide primer according to (1) comprising at least 15continuous nucleotides selected from the nucleotide sequences as shownin SEQ ID NOs: 2 to 13 selected from the nucleotide sequence of a RNApolymerase of the SARS coronavirus or a nucleotide sequencecomplementary thereto.

(3) The oligonucleotide primer according to (1) or (2) consisting of thenucleotide sequence selected from the following nucleotide sequences (a)to (d), provided that nucleotide sequence regions F3c, F2c, and F1c areselected from the 3′-terminus and nucleotide sequence regions R3, R1,and R1 are selected from the 5′-terminus of the target nucleic acid ofthe SARS coronavirus, and nucleotide sequences complementary thereto aredetermined to be F3, F2, and F1 and R3c, R2c, and R1c, respectively:

(a) a nucleotide sequence having the F2 region and the F1c region of thetarget nucleic acid at the 3′-terminus and the 5′-terminus,respectively;

(b) a nucleotide sequence having the F3 region of the target nucleicacid;

(c) a nucleotide sequence having the R2 region and the R1c region of thetarget nucleic acid at the 3′-terminus and the 5′-terminus,respectively; and

(d) a nucleotide sequence having the R3 region of the target nucleicacid.

(4) The oligonucleotide primer according to any of (1) to (3) capable ofamplifying a SARS coronavirus-specific nucleotide sequence andconsisting of a nucleotide sequence selected from the following (e) to(h) from the 5′-terminus toward the 3′-terminus:

(e) 5′-(a nucleotide sequence complementary to the nucleotide sequenceas shown in SEQ ID NO: 2)-(any nucleotide sequence comprising 0 to 50nucleotides)-(the nucleotide sequence as shown in SEQ ID NO: 3)-3′;

(f) 5′-(the nucleotide sequence as shown in SEQ ID NO: 5)-(anynucleotide sequence comprising 0 to 50 nucleotides)-(a nucleotidesequence complementary to the nucleotide sequence as shown in SEQ ID NO:6)-3′;

(g) 5′-(a nucleotide sequence complementary to the nucleotide sequenceas shown in SEQ ID NO: 8)-(any nucleotide sequence comprising 0 to 50nucleotides)-(the nucleotide sequence as shown in SEQ ID NO: 9)-3′; and

(h) 5′-(the nucleotide sequence as shown in SEQ ID NO: 11)-(anynucleotide sequence comprising 0 to 50 nucleotides)-(a nucleotidesequence complementary to the nucleotide sequence as shown in SEQ ID NO:12)-3′.

(5) A method for detecting the SARS coronavirus comprising amplifying atarget nucleic acid region of the SARS coronavirus using theoligonucleotide primer according to any of (1) to (4).

(6) The method according to (5), wherein a target nucleic acid region ofthe SARS coronavirus is amplified by the LAMP method.

(7) A method for diagnosing severe acute respiratory syndrome (SARS)comprising diagnosing infection with the SARS coronavirus by detectingamplification of a target nucleic acid region of the SARS coronavirususing the oligonucleotide primer according to any of (1) to (4).

(8) A kit used for a method for diagnosing severe acute respiratorysyndrome (SARS) comprising the oligonucleotide primer according to anyof (1) to (4).

EFFECT OF THE INVENTION

According to the present invention, an oligonucleotide primer that canselectively hybridize with a SARS coronavirus-specific nucleotidesequence is produced, and a SARS coronavirus-specific nucleotidesequence is amplified by the LAMP method. Thus, the SARS coronavirus canbe detected with high sensitivity and rapidity.

Hereafter, the present invention is described in detail.

Preferred Embodiments of the Invention

Samples that are used in the present invention are specimens obtainedfrom humans or other animals suspected of having SARS. Examples thereofinclude sputum, bronchoalveolar lavage fluid, rhinorrhea, nasalaspirate, nasal wash, nasal sponge, pharyngeal sponge, mouth washing,saliva, blood, serum, blood plasma, spinal fluid, urine, stool, andtissue. In addition, specimens including, for example, cells used forinfection experiments or the like, a culture solution thereof, orviruses separated from specimens obtained from organisms or culturedcells can be employed as samples. Such samples may be subjected topretreatment such as separation, extraction, concentration, orpurification.

Nucleic acids can be amplified by a novel technique of nucleic acidamplification that is referred to as the loop-mediated isothermalamplification (LAMP) method (WO 00/28082). This LAMP method wasdeveloped by Notomi et al. and eliminated the need for temperaturecontrol, which is indispensable for PCR. In this method, the 3′terminuses of template nucleotides are annealed, synthesis ofcomplementary strands is started therefrom, and a primer that isannealed to the loop formed via the aforementioned synthesis is used incombination therewith. This enables nucleic acid amplification to becarried out under isothermal conditions. In the LAMP method, the 3′terminus of the primer is always annealed to a sample-derived region,and thus, a mechanism for checking upon complementary bonding ofnucleotide sequences functions repeatedly. Consequently, nucleic acidamplification with high sensitivity and specificity is realized.

In the LAMP reaction, at least 4 types of oligonucleotide primers areused. These primers recognize a total of 6 regions in the nucleotidesequence of the template nucleic acid, i.e., the nucleotide sequences ofF3c, F2c, and F1c regions from the 3′ terminus and R3, R2, and R1regions from the 5′ terminus. These primers are referred to as innerprimers F and R and outer primers F and R. The complementary sequencesof F1c, F2c, and F3c are referred to as F1, F2, and F3, respectively,and the complementary sequences of R1, R2, and R3 are referred to asR1c, R2c, and R3c, respectively. An inner primer is an oligonucleotidethat recognizes a “given nucleotide sequence region” on the targetnucleotide sequence. It has on its 3′ terminus the nucleotide sequenceof the synthesis origin, and it has on its 5′ terminus a nucleotidesequence complementary to any region of the product of nucleic acidsynthesis originating from this primer. In the present invention, aprimer comprising a “nucleotide sequence selected from F2” and a“nucleotide sequence selected from F1c” is referred to as an “innerprimer F (hereafter abbreviated as “IPF”),” and a primer comprising a“nucleotide sequence selected from R2” and a “nucleotide sequenceselected from R1c” is referred to as an “inner primer R (hereafterabbreviated as “IPR”).” In contrast, an outer primer is anoligonucleotide that recognizes “an arbitrary nucleotide sequence regionlocated on the 3′ terminal side of a ‘given nucleotide sequenceregion’,” and a nucleotide sequence serving as an origin of synthesis onthe target nucleotide sequence. In the present invention, a primercomprising a “nucleotide sequence selected from F3” is referred to as an“outer primer F (hereafter abbreviated as “OPF”),” and a primercomprising a “nucleotide sequence selected from R3” is referred to as an“outer primer R (hereafter abbreviated as “OPR”).” “F” in each primerindicates a primer that complementarily binds to a sense strand of thetarget nucleotide sequence and functions as a synthesis origin. “R”indicates a primer that complementary binds to an antisense strand ofthe target nucleotide sequence and functions as a synthesis origin. Thelength of the oligonucleotide used as a primer is at least 10nucleotides, and preferably at least 15 nucleotides. It may bechemically synthesized or naturally occurring. Each primer may be asingle oligonucleotide or a mixture of a plurality of oligonucleotides.

In the LAMP method, another primer, i.e., a loop primer, can be used inaddition to the inner and the outer primers. A loop primer has anucleotide sequence that is complementary to a nucleotide sequence in asingle-stranded region of the loop structure on the 5′ terminal side ofa dumbbell structure. With the use of such primer, the number of originsof nucleic acid synthesis can be increased, the reaction time can beshortened, and the detection sensitivity can be improved (WO 02/24902).The nucleotide sequence of the loop primer may be selected from thenucleotide sequence of the target gene or a complementary strandthereof. Alternatively, it may be another nucleotide sequence as long asit is complementary to the nucleotide sequence in the single-strandregion in the loop structure on the 5′ terminal side of theaforementioned dumbbell structure. A single type or two or mote types ofloop primers may be used.

The SARS coronavirus is an RNA virus. When an RNA template is employedin the LAMP method, nucleic acid can be similarly amplified as with thecase of a reaction using a DNA template by using a reaction solutionprepared by adding a reverse transcriptase to the reaction solution forthe latter type of reaction (the RT-LAMP method).

The present inventors have thoroughly studied the nucleotide sequencesof primers for the LAMP method that can rapidly amplify a SARScoronavirus-specific nucleotide sequence and combinations thereof. As aresult, they selected the following primer sets A and B based on thenucleotide sequence as shown in SEQ ID NO: 1 from the nucleotidesequence of an RNA polymerase of the SARS coronavirus (Drosten C., etal., New Eng. J. Med., 2003, vol. 348, pp. 1967-1976).

(Primer set A) IPF-A: 5′-TACATCAAAGCCAATCCACGCAATATGTTTATCACCCGCGAAGA-3′(SEQ ID NO: 14) OPF-A: 5′-ACCAAGTCAATGGTTACCCT-3′ (SEQ ID NO: 4) IPR-A:5′-GCTGTCATGCAACTAGAGATGCTACAGCTACTAAGTTAACACCTG-3′ (SEQ ID NO: 15)OPR-A: 5′-GTGTCAACATAACCAGTCGG-3′ (SEQ ID NO: 16) LPF-A:5′-ACGAACGTGACGAATAGCT-3′ (SEQ ID NO: 20) LPR-A:5′-GTACTAACCTACCTCTCCAGC-3′ (SEQ ID NO: 21) (Primer set B) IPF-B:5′-TGCATGACAGCCCTCGAAGAAGCTATTCGTCAC-3′ (SEQ ID NO: 17) OPF-B:5′-CTAATATGTTTATCACCCGC-3′ (SEQ ID NO: 10) IPR-B:5′-GCTGTGGGTACTAACCTACCTGTCAACATAACCAGTCGG-3′ (SEQ ID NO: 18) OPR-B:5′-CTCTGGTGAATTCTGTGTT-3′ (SEQ ID NO: 19) LPF-B: 5′-AAAGCCAATCCACGC-3′(SEQ ID NO: 22) LPR-B: 5′-CCAGCTAGGATTTTCTACAGG-3′ (SEQ ID NO: 23)

Any template-dependent nucleic acid synthetases having stranddisplacement activity can be used for nucleic acid synthesis withoutparticular limitation. Examples of such enzymes include Bst DNApolymerase (large fragment), Bca(exo-) DNA polymerase, and the Klenowfragment of E. coli DNA polymerase I. Bst DNA polymerase (largefragment) is preferable.

Any enzyme having activity of synthesizing DNA with the use of an RNAtemplate can be used as a reverse transcriptase for the RT-LAMP methodwithout particular limitation. Examples of such enzyme include reversetranscriptase, such as AMV, Cloned AMV, MMLV, SuperscriptII,ReverTraAce, and Thermoscript. Reverse transcriptase, such as AMV orCloned AMV, is preferable. With the use of an enzyme having activity ofreverse transcriptase and of DNA polymerase, such as Bca DNA polymerase,the RT-LAMP reaction can be carried out using a single enzyme.

An enzyme or reverse transcriptase that is used for nucleic acidsynthesis may be purified from a virus or bacterium, or it may beprepared via gene recombination. Alternatively, such enzyme may besubjected to modification such as fragmentation or amino acidsubstitution.

After the LAMP reaction, a product of nucleic acid amplification can bedetected via a conventional technique. For example, such product can beeasily detected by using a labeling oligonucleotide that specificallyrecognizes an amplified nucleotide sequence or a fluorescentintercalator-based method (JP Patent Publication (Kokai) No. 2001-242169A) or by subjecting the reaction solution after the completion of thereaction to agarose gel electrophoresis. The product of LAMPamplification is detected as a ladder of multiple bands of differentnucleotide lengths via agarose gel electrophoresis. In the LAMP method,a large quantity of substrates are consumed upon nucleic acid synthesis,pyrophosphoric acid as a by-product is converted into magnesiumpyrophosphate upon reaction with magnesium, which is also presenttherein, and the reaction solution becomes turbid to the extent suchthat such turbidity can be visually inspected. Accordingly, nucleic acidamplification can be detected with the elapse of time by observing suchturbidity using a measuring apparatus that allows optical observation ofthe level of turbidity after the completion of a reaction or during areaction, for example, measuring changes in absorbance at 400 nm using ageneral spectrophotometer (WO 01/83817).

Any of a variety of reagents that are necessary for detecting nucleicacid amplification with the use of the primer according to the presentinvention can serve as a pre-packaged kit. More specifically, the kitcomprises various types of oligonucleotides that are necessary asprimers according to the present invention or the loop primers, 4 typesof dNTP that are substrates for nucleic acid synthesis, DNA polymerasefor nucleic acid synthesis, an enzyme having activity of reversetranscriptase, a buffer or salts providing suitable conditions forenzyme reactions, a protecting agent for stabilizing enzymes ortemplates, and reagents necessary for detecting reaction products,according to need.

EXAMPLES

The present invention is hereafter described in greater detail withreference to the following examples, although the present invention isnot limited thereto.

Example 1 Confirmation of Detection Sensitivity

Sensitivity of LAMP detection was compared with that of PCR detection.

1. Preparation of Samples and Reagents

1) Samples

RNA as shown in SEQ ID NO: 1 selected from RNA polymerase sequences ofthe SARS coronavirus was dissolved in a yeast RNA solution (50 ng/μl,Ambion), a dilution containing 10 to 10⁴ copies of RNA per μl anddilutions containing 2.5, 5, and 10 copies thereof were prepared, andthese dilutions were designated as sample solutions. The aforementionedyeast RNA solution was designated as a sample solution containing 0copies.

2) Composition and Concentration of Reagent Used for PCR

PCR was carried out in accordance with the method described in thenon-patent document 4, wherein two types of primers each independentlyconsisting of the nucleotide sequences as shown in SEQ ID NOs: 24 and 25that amplify a 195-bp fragment of RNA polymerase were used as primersfor detecting the SARS coronavirus.

Composition of cDNA Synthesis Reaction Solution

-   -   4 μL of 5× first strand buffer (Invitrogen)    -   1L of 10 mM dNTPs    -   2 L of 0.1M DTT    -   1 μL of random primer (50 ng/μl, TAKARA)    -   1 μL of RNase inhibitor (40 U/μl, Invitrogen)    -   1 μL of SuperScriptII (200 U/μl, Invitrogen)    -   5 μL of distilled water    -   5 μL of sample solution        Composition of PCR Solution    -   5 μL of 10×Ex Taq buffer (Mg free) (TAKARA)    -   4 L of 25 mM MgCl₂    -   4 L of 2.5 mM dNTPs    -   1 μL each of 10 μmol/μL primer    -   0.5 μL of Ex Taq (5 U/μL, TAKARA)    -   33.5 μL of distilled water    -   1 μL of cDNA synthesis solution        3) Composition and Concentration of Reagent for the LAMP Method

The concentrations of reagents in 25 μl of the final reaction solutionwere adjusted to the following levels for LAMP amplification with theuse of the primer set A.

Composition of Reaction Solution

-   -   20 mM Tris-HCl (pH 8.8)    -   10 mM KCl    -   8 mM MgSO₄    -   1.4 mM dNTPs    -   10 mM (NH₄)₂SO₄    -   0.8 M Betaine (Sigma)    -   0.1% Tween 20    -   1.6 μM IPF and IPR    -   0.2 μM OPF and OPR    -   0.8 M LPF and LPR    -   0.625 U of AMV reverse transcriptase (Invitrogen)    -   8 U of Bst DNA polymerase (New England Biolabs)    -   0.25 μg/mL EtBr (Nippon Gene Co., Ltd.)

In the case of a reaction where the primer set B was used, 2 U of ClonedAMV reverse transcriptase (Invitrogen) was used instead of 0.625 U ofAMV reverse transcriptase.

2. Reaction Via Nucleic Acid Amplification

1) Reaction Via PCR

A sample solution (5 μl) containing 0 or 10 to 10³ copies of the targetsequences was added to the aforementioned cDNA synthesis solution, andthe resulting mixture was subjected to cDNA synthesis at 42° C. for 50minutes and then at 70° C. for 15 minutes. A solution of synthesizedcDNA (1 μl) was added to the aforementioned PCR solution to bring thefinal amount thereof to 50 μl, and the reaction solution was subjectedto PCR in a 0.2-ml dedicated purpose tube using a PTC-200 thermal cycler(MJ Research). A cycle of denaturation at 95° C. for 30 seconds,annealing at 56° C. for 30 seconds, and polymerase elongation at 72° C.for 30 seconds was repeated 40 times. The time required for completingPCR was approximately 1 hour. After the completion of the reaction, 5 μlof the reaction solution was subjected to 2% agarose gelelectrophoresis.

2) Reaction Via LAMP

A sample solution (1 μl) containing 0 or 10 to 10³ copies of the targetsequences was added to a reagent for LAMP using the primer set A tobring the final amount thereof to 25 μl, and the reaction solution wassubjected to LAMP in a 0.2-ml dedicated purpose tube at 63° C. for 60minutes. After the completion of the reaction, 5 μl of the reactionsolution was subjected to 2% agarose gel electrophoresis.

3. Result of Comparing Sensitivity for Detecting Each Nucleic AcidAmplification Product Via Electrophoresis

FIG. 1 shows the results of observing the detection sensitivity of PCRby electrophoresis, and FIG. 2 shows the results of observing thedetection sensitivity of LAMP using the primer set A by electrophoresis.As a result, amplification products were observed both in PCR and inLAMP. In the case of PCR, a 195-bp specific band was clearly observed ina dilution containing 10² copies; however, the amplification product wasobserved as an unclear band in the case of a dilution containing 10copies. In contrast, a specific amplification product was observed as aladder-like band in a dilution containing only 10 copies in the case ofLAMP.

Example 2 Determination of Time Required for Real-time LAMP Detection

The time required for LAMP detection with the use of the primer set Awas examined using 25 μl of the composition for the LAMP method inExample 1, using a real-time fluorescent measuring apparatus (PRISM7700, Applied Biosystems), and fixing the reaction temperature at 63° C.The results are shown in FIG. 3.

As a result, no increase in fluorescence was observed in a samplecontaining 0 copies 60 minutes later. In contrast, an increase influorescence was observed in a sample containing 10 copies or morewithin 20 minutes. This indicates that 10 copies were detected within 20minutes.

The time required for LAMP detection with the use of the primer set Bwas examined via real-time turbidimetry using a real-time turbiditymeasuring apparatus (LA-200, Teramecs Co., Ltd.). The LAMP reaction wascarried out using the LAMP composition (25 μl) prepared in Example 1 andfixing the reaction temperature at 63° C. The results are shown in FIG.4.

As a result, no increase in turbidity was observed in a samplecontaining 0 copies 60 minutes later. In contrast, an increase inturbidity was observed in a sample containing 2.5 copies or more within35 minutes. This indicates that 2.5 copies were detected within 35minutes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows sensitivity of PCR detection observed by electrophoresis(lanes 1 and 7: markers; lane 2: a reagent blank; lane 3: 0 copies; lane4: 10 copies; lane 5: 10² copies; and lane 6: 10³ copies).

FIG. 2 shows sensitivity of LAMP detection using the primer set Aobserved by electrophoresis (lanes 1: 0 copies; lane 2: 10 copies; lane3: 10² copies; lane 4: 10³ copies; lane 5: 10⁴ copies; and lane 6: amarker).

FIG. 3 shows the detection time of the real-time fluorescence assayusing the primer set A.

FIG. 4 shows the detection time of the real-time turbidimetry using theprimer set B.

1. A primer set for detecting SARS coronavirus comprising: a) a firstoligonucleotide primer comprising SEQ ID NO: 17 or a nucleotide sequenceentirely complementary to said first oligonucleotide primer, b) a secondoligonucleotide primer comprising SEQ ID NO: 18 or a nucleotide sequenceentirely complementary to said second oligonucleotide primer, c) a thirdoligonucleotide primer comprising at least 15 contiguous nucleotides ofSEQ ID NO: 10 or a nucleotide sequence entirely complementary to saidthird oligonucleotide primer, and d) a fourth oligonucleotide primercomprising at least 15 contiguous nucleotides of SEQ ID NO: 19 or anucleotide sequence entirely complementary to said fourtholigonucleotide primer.
 2. The primer set of claim 1, further comprisinga) a fifth oligonucleotide primer comprising at least 15 contiguousnucleotides of SEQ ID NO: 22, or a nucleotide sequence entirelycomplementary to said fifth oligonucleotide primer, and b) a sixtholigonucleotide primer comprising at least 15 contiguous nucleotides ofSEQ ID NO: 23, or a nucleotide sequence entirely complementary to saidsixth oligonucleotide primer.
 3. A kit used for a method for detectingsevere acute respiratory syndrome (SARS) comprising the primer set ofclaim
 1. 4. The kit of claim 3, further comprising a) a fiftholigonucleotide primer comprising at least 15 contiguous nucleotides ofSEQ ID NO: 22, or a nucleotide sequence entirely complementary to saidfifth oligonucleotide primer, and b) a sixth oligonucleotide primercomprising at least 15 contiguous nucleotides of SEQ ID NO: 23, or anucleotide sequence entirely complementary to said sixth oligonucleotideprimer.